Numerous genetic diseases are caused by mutations in the mammalian genome. Other sources of genetic diseases are activation of silent genes or the presence of viral genes in the mammalian genome. Several types of modifications have been found to be mutated in the genome: deletion of one or several base pairs, one or several mismatches in the sequence of the gene, insertion of one or several bases, or repeat triplet reiteration and absence of a whole or part of a gene.
Genetic diseases caused by mismatches, deletion/insertion of one or several base pairs (BP) and repeat triplet mutation in the genes include albinism, cystic fibrosis, muscular dystrophy and atrophy, sickle cell anemia, hepatic disorders, hemophilia, Crigler-Najjar syndrome, renal tubular acidosis, β-thalassemia, atherosclerosis, Huntington's disease, spinocerebellar ataxia (type 1, 2 and 6), Machado-Joseph disease, myotonic dystrophy, Fragile X (forms A and B) and Frederich's ataxia [Breschel et al., Human Molec. Gen. (1997) 6, 1855-1863; Kmiec, Clin. Invest. (2003) 112, 632-636].
Oligonucleotide complexes and their analogs have been employed as a potential therapeutic for the readout of genes [McManus et al., Nat Rev Genet. (2002) 3, 737-747; Nielsen, Curr. Med. Chem. (2001) 8, 545-550; Agrawal et al., Curr. Cancer Drug Targets. (2001) 1, 197-209] and for targeted gene repair [Kmiec, Clin. Invest. (2003) 112, 632-636].
An approach in the field of gene therapy is introduction of sequence-specific modification of the genes, using oligonucleotide complexes for the phenotypic and/or genotypic restoration of defective genes. Approaches to an oligonucleotide-based strategy to achieve this goal have been tested. Chimeric RNA/DNA oligonucleotides and single-stranded oligonucleotides were developed for site-specific correction of episomal and chromosomal target genes [Andersen et al. J Mol Med. (2002) 80, 770-81; Alexeev et al., Gene therapy (2002) 9, 1667-1675; Kmiec, Clin. Invest. (2003) 112, 632-636; Wu et al., J Biomed Sci. (2001) 8, 439-45; Yoon, U.S. Patent Application Publication 1999000473872; Davis et al., U.S. Patent Application Publication 2000767775; Youn et al., U.S. Patent Application Publication 2001000962628; Kmiec et al., U.S. Patent Application Publication 2002000260375; Kmiec et al., U.S. Patent Application Publication 2002000215432]. Experiments demonstrated the feasibility of using chimeric RNA/DNA and single stranded oligonucleotides to introduce point conversions in genes in vitro and in vivo. This gene repair approach relies on hybridization of the chimera to the target gene, generating a mismatch with the targeted point mutation. Restored gene function was anticipated to occur through activation of endogenous repair systems that recognize the created mismatch [Andersen et al., J Mol Med. (2002) 80, 770-81; Alexeev et al., Gene therapy (2002) 9, 1667-1675; Kmiec, Clin. Invest. (2003) 112, 632-636; Wu et al., J Biomed Sci. (2001) 8, 439-45; Wang et al., (2003) Proc. Natl. Acad. Sci. USA 100, 14822-14827]. Double stranded oligonucleotides have been tested for site specific gene alteration in plant cells [Arntzen et al., U.S. Patent Application Publication 1998000129298; Kmiec, U.S. Patent Application Publication 1994000353657].
Triplex forming oligonucleotides also have been employed as sequence-specific tools for gene targeting. Triplex forming oligonucleotides bind in the major groove of double stranded DNA, with high affinity. Because of this characteristic, triplex forming oligonucleotides have been proposed as tools for the site specific corrections of targeted genes [Knauert et al., Hum Mol. Genet. (2001) 10, 2243-2251; Richardson et al., Drug Target (2002) 10, 133-134; Thoung et al., (1993) Angewandte Chemie. Intl. Ed. Eng., 32, 666-690.].
Current targeted gene repair methods are controversial and still at the level of development. There is a need for more effective tools, in order to obtain phenotypic or genotypic restoration of defective genes in somatic tissues.